Mécanismes moléculaires d'adaptation aux conditions physico-chimiques extrêmes dans la famille des lactate-malate déshydrogénases

La vie est présente partout, ou presque, sur Terre, et notamment dans des environnements considérés comme extrêmes. Ces organismes extrêmophiles, non contents de subsister sous ces contraintes physico-chimiques extrêmes, s'y complaisent. Plusieurs mécanismes adaptatifs, à divers niveaux de l'organisation cellulaire, ont été mis en place. Les études présentées ici s'intéressent aux mécanismes d'adaptation moléculaire des protéines en utilisant la famille des lactate-malate déshydrogénases comme modèle. Il semblerait qu'une réorganisation des interactions au sein de la structure assure la stabilité, la solubilité et l'activité de la protéine sous cette contrainte physico-chimique extrême.Dans une première partie, les propriétés biochimiques et structurales des lactates déshydrogénases de la bactérie thermophile Thermus thermophilus (TtLDH1 de la bactérie mésophile Deinococcus radiodurans (DrLDH) et du poisson psychrophile de Champsocepha/us gunnari (CgLDH) ont été déterminées. Les deux premières ont été comparées pour étudier la transition thermophile/mésophile. La dernière a été comparée à la LDH de Squa/us acanthias (SaLDH) pour comprendre la transition mésophile/psychrophile. Peu de substitutions semblent être à l'origine des différences de propriétés thermiques entre ces enzymes. Cette hypothèse a pu être vérifiée par la caractérisation d'un mutant de TtLDH. Cinq substitutions judicieusement choisies ont permis d'altérer ses propriétés thermiques.L'adaptation moléculaire aux fortes concentrations en sel a été, pendant longtemps, étudiée en utilisant la malate désydrogénase de Ha/oarcu/a marismortui (HmMalDH). Nous présentons ici les caractéristiques biochimiques et structurales de la malate désydrogénase issue de Sa/inibacter ruber (SrMaIDH), la seule bactérie halophile extrême connue à ce jour. Les propriétés de cette enzyme semblent intermédiaires entre une enzyme non halophile et une enzyme totalement efficace aux fortes concentrations en sel. Cela a permis, pour la première fois, de proposer un découplage des différents effets affectant la stabilité conformationnelle,l'activité enzymatique et la solubilité. Enfin, l'irradiation des cristaux de protéine provoque des dommages au sein de la structure de la macromolécule, et touche notamment les groupements carboxyle des chaînes latérales des résidus acides. La forme apo (enzyme seule) et le complexe ternaire (enzyme :NADH :analogue de substrat) de TtLDH ont été irradiés et, à dose absorbée équivalente, l'amplitude des dommages entre les deux formes a été comparée. La fixation du cofacteur et du substrat ne protège pas des radiations le résidu acide présent dans le site actif.Life is found everywhere, or almost, on earth and particularly in environments considered as extreme. These extremophilic organisms do not only subsist, but thrive in these conditions. Several adaptative mechanisms, at different cellular levels, have beendevelopped. Our studies focus on molecular mechanisms of proteins adaptation, using the lactate-malate dehydrogenases family as model. An intramolecular reorganization of interactions seems to be sufficient to en sure conformational stability, enzymatic activity and solubility of the protein in the se eXtreme conditions.First, biochemical and structural properties of lactate dehydrogenases from the thermophilic bacterium Thermus thermophilus (TtLDH), the mesophilic bacterium Deinococcus radiodurans (DrLDH) and the psychrophilic fish Champsocepha/us gunnari (CgLDH) have been determined. The first two have been compared to understand the thermophilic/mesophilic transition. The latter has been compared to the LDH from Squa/us acanthias (SaLDH) to study the mesophilic/psychrophilc transition. Few substitutions seems to be responsible for the differences in thermal properties between these enzymes. This hypothesis has been validated using a molecular variant of TtLDH. Five amino acid substitutions have modified its thermal properties.Molecular adaptation to high salt concentrations has been studied for a long time using the Ha/oarcu/a marismortui malatedehydrogenase (HmMaIDH). We present here the biochemical and structural properties of the malate dehydrogenase tTom Sa/inibacter ruber (SrLDH), the only extreme halophilic bacterium known to date. Properties of this enzyme appear to be 'intermediate' between a non halophilic enzyme and a fully active enzyme in high salt. For the first time, different effects that modiry conformational stability, enzymatic activity or solubility have been uncoupled.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Three-dimensional structure of quinoprotein methylamine dehydrogenase.

In this thesis, the crystal structure determination of quinoprotein methylamine dehydrogenase to a resolution of 2.25 À is described. The structure of this enzyme, complete with its PQQ-related quinone cofactor is presented, and relevant observations concerning this structure are discussed. ... Zie: Summary

Methanoarchaeal sulfolactate dehydrogenase: prototype of a new family of NADH-dependent enzymes

The crystal structure of the sulfolactate dehydrogenase from the hyperthermophilic and methanogenic archaeon Methanocaldococcus jannaschii was solved at 2.5 Å resolution (PDB id. 1RFM). The asymmetric unit contains a tetramer of tight dimers. This structure, complexed with NADH, does not contain a cofactor-binding domain with ‘Rossmann-fold' topology. Instead, the tertiary and quaternary structures indicate a novel fold. The NADH is bound in an extended conformation in each active site, in a manner that explains the pro-S specificity. Cofactor binding involves residues belonging to both subunits within the tight dimers, which are therefore the smallest enzymatically active units. The protein was found to be a homodimer in solution by size-exclusion chromatography, analytical ultracentrifugation and small-angle neutron scattering. Various compounds were tested as putative substrates. The results indicate the existence of a substrate discrimination mechanism, which involves electrostatic interactions. Based on sequence homology and phylogenetic analyses, several other enzymes were classified as belonging to this novel family of homologous (S)-2-hydroxyacid dehydrogenases

Using lanthanoid complexes to phase large macromolecular assemblies

International audienc

Crystal structure of pb9, the distal tail protein of bacteriophage T5: a conserved structural motif among all siphophages.

International audienceThe tail of Caudovirales bacteriophages serves as an adsorption device, a host cell wall-perforating machine, and a genome delivery pathway. In Siphoviridae, the assembly of the long and flexible tail is a highly cooperative and regulated process that is initiated from the proteins forming the distal tail tip complex. In Gram-positive-bacterium-infecting siphophages, the distal tail (Dit) protein has been structurally characterized and is proposed to represent a baseplate hub docking structure. It is organized as a hexameric ring that connects the tail tube and the adsorption device. In this study, we report the characterization of pb9, a tail tip protein of Escherichia coli bacteriophage T5. By immunolocalization, we show that pb9 is located in the upper part of the cone of the T5 tail tip, at the end of the tail tube. The crystal structure of pb9 reveals a two-domain protein. Domain A exhibits remarkable structural similarity with the N-terminal domain of known Dit proteins, while domain B adopts an oligosaccharide/oligonucleotide-binding fold (OB-fold) that is not shared by these proteins. We thus propose that pb9 is the Dit protein of T5, making it the first Dit protein described for a Gram-negative-bacterium-infecting siphophage. Multiple sequence alignments suggest that pb9 is a paradigm for a large family of Dit proteins of siphophages infecting mostly Gram-negative hosts. The modular structure of the Dit protein maintains the basic building block that would be conserved among all siphophages, combining it with a more divergent domain that might serve specific host adhesion properties

An archaeal peptidase assembles into two different quaternary structures: A tetrahedron and a giant octahedron.

International audienceCellular proteolysis involves large oligomeric peptidases that play key roles in the regulation of many cellular processes. The cobalt-activated peptidase TET1 from the hyperthermophilic Archaea Pyrococcus horikoshii (PhTET1) was found to assemble as a 12-subunit tetrahedron and as a 24-subunit octahedral particle. Both quaternary structures were solved by combining x-ray crystallography and cryoelectron microscopy data. The internal organization of the PhTET1 particles reveals highly self-compartmentalized systems made of networks of access channels extended by vast catalytic chambers. The two edifices display aminopeptidase activity, and their organizations indicate substrate navigation mechanisms different from those described in other large peptidase complexes. Compared with the tetrahedron, the octahedron forms a more expanded hollow structure, representing a new type of giant peptidase complex. PhTET1 assembles into two different quaternary structures because of quasi-equivalent contacts that previously have only been identified in viral capsids

Mutations in GFPT1-related congenital myasthenic syndromes underlie a tubular aggregates myopathy with synaptopathy

International audienceMutations in GFPT1 (glutamine-fructose-6-phosphate transaminase 1), a gene encoding an enzyme involved in glycosylation of ubiquitous proteins, cause a limb-girdle congenital myasthenic syndrome (LG-CMS) with tubular aggregates (TAs) characterized predominantly by affection of the proximal skeletal muscles and presence of highly organized and remodeled sarcoplasmic tubules in patients’ muscle biopsies. GFPT1 is the first and rate-limiting enzyme of the hexosamine biosynthetic pathway (HBP) involved in ubiquitous glycosylation processes (Senderek J. et al., 2011). GFPT1 is the primary genetic cause of ubiquitous CMS. Since 2011, a total of 52 patients with GFPT1 mutations have been clinically reported (Guergueltcheva V. et al., 2012; Senderek et al., 2011;Selcen D. et al., 2013; Huh S-Y. et al., 2012). Interestingly, GFPT1 is expressed in various tissues, including skeletal muscle and motor nerve, raising the intriguing question of why mutations in such a ubiquitous gene would specifically lead to synaptic and muscular disorganization. We report here the first retrospective clinical description and the molecular investigations of 11 individuals with LG-CMS linked to recessively-inherited mutations in GFPT1.In this study, we report the first long-term clinical follow-up of 11 French individuals from 9 unrelated families suffering from LG-CMS with TAs due to GFPT1 mutations, of which 9 are new. Our retrospective clinical evaluation stresses an evolution toward a myopathic weakness that occurs concomitantly to ineffectiveness of usual CMS treatments. Analysis of neuromuscular biopsies from 3 unrelated individuals demonstrates that the maintenance of neuromuscular junctions (NMJs) is dramatically impaired with loss of post-synaptic junctional folds and evidence of denervation-reinnervation processes affecting the 3 main NMJ components. Moreover, molecular analyses of the human muscle biopsies confirm glycosylation defects of proteins with reduced O-glycosylation and show reduced sialylation of transmembrane proteins in extrajunctional area. Altogether, these results pave the way for understanding the etiology of this rare neuromuscular disorder that may be considered as a “tubular aggregates myopathy with synaptopathy